1. Field of the invention
The present invention generally relates to a process for enzymatic replacement of the C-terminal amino acid in the B-chain (B-30) of insulins from various species.
It is well known that insulins from different vertebrate species including mammals and humans differ in their primary structure. Since Sanger in 1958 determined the primary structure of bovine insulin the primary structure of insulins from other vertebrate species has been determined.
These results as summarized in the below figure using the porcine insulin as model indicate that amino acid substitutions can occur at many positions within either chain. However, certain structural features are common to all the insulins, e.g. the position of the 3 disulfide bonds, the N-terminal region of the A-chain, the B23-26 sequence in the C-terminal region of the B-chain, etc.
The differences in the primary structure of some common insulins are seen from the below table:
______________________________________ A-chain B-chain 4 8 9 10 3 29 30 ______________________________________ Bovine Glu Ala Ser Val Asn Lys Ala Sheep Glu Ala Gly Val Asn Lys Ala Horse Glu Thr Gly Ileu Asn Lys Ala Sei whale Glu Ala Ser Thr Asn Lys Ala Porcine Glu Thr Ser Ileu Asn Lys Ala Sperm whale Glu Thr Ser Ileu Asn Lys Ala Dog Glu Thr Ser Ileu Asn Lys Ala Human Glu Thr Ser Ileu Asn Lys Thr Rabbit Glu Thr Ser Ileu Asn Lys Ser Rat 1 Asp Thr Ser Ileu Lys Lys Ser Rat 2 Asp Thr Ser Ileu Lys Met Ser ______________________________________
While the invention is described more fully below with relation to the specific conversion of porcine insulin into human insulin, viz. replacement of B-30 alanine by threonine, it will easily be understood that the described method applies equally well to other types of insulin in that e.g. rabbit insulin may also be converted into human insulin, bovine insulin may be converted into B-30 (Thr) bovine insulin, etc.
2. Background of invention, especially with relation to conversion of porcine insulin into human insulin
The idea of converting porcine insulin into human insulin by semi-synthetic procedure has been an attractive problem in the field of insulin chemistry.
As stated above, human insulin differs from porcine insulin by only one amino acid, the C-terminal residue of the B-chain (B-30) being threonine in human and alanine in porcine insulin, respectively. The exchange of alanine to threonine was initially performed chemically and recently enzymatic procedures have been used. Ruttenberg (1972) (Ref. 1) has described the chemical conversion of porcine insulin into human insulin: esterification to insulin-hexamethylester, hydrolysis with trypsin to desoctapeptide insulin (DOI)-pentamethylester, blocking of the amino terminal residues, chemical coupling with a synthetic octapeptide of the corresponding human insulin sequence, acidic deprotection of the amino-groups, and finally alkaline saponifcation of the methyl ester groups. However, nobody has ever been able to produce pure human insulin by this method, since the chemical procedures, and in particular the final alkaline saponification steps seriously damage the insulin molecule and also isoasparagine at the C-terminal residue of the A-chain is formed (Gattner et al. (Ref. 2)). To prevent this effect Obermeier and Geiger (1976) (Ref. 3) have carried out the fragment condensation without protection of the side chain carboxyl groups of DOI. They could isolate human insulin after extensive purification, but only in very low yields. Similar approaches have been taken by Gattner et al. (Ref. 2), using various insulin fragments. However, using the chemical methods nobody has so far prepared pure human insulin in more than trace amounts.
M. Bodanszky et al. provides a process for preparing human insulin in U.S. Pat. No. 3,276,961 wherein human insulin was ostensibly prepared from other animal insulins by an action of an enzyme such as carboxypeptidase A and trypsin in the presence of threonine. This process is not likely to produce human insulin because trypsin and carboxypeptidase A hydrolyze not only the peptide bond of lysyl-alanine (B29-B30) but also the other positions in insulin under the condition described there. Trypsin preferentially hydrolyzes the peptide bond of arginyl-glycine (B22-B23) than that of lysyl-alanine (B29-B30). Meanwhile, carboxypeptidase A cannot release solely the alanine at C-terminal of the B chain without liberating asparagine at C-terminal of the A chain. A special condition, i.e. reacting in an ammonium hydrogencarbonate buffer solution, is necessary to prevent the release of the asparagine. The condition was discovered in 1978 (Schmitt, Hoppe-Seyler's Z. Physiol. Chem., 359, 799-802 (1978)). Furthermore, peptide synthesis may hardly occur because hydrolysis ratio is faster than synthesis ratio in the condition. Inouye et al. (Ref. 4) have shown that human insulin can be obtained by coupling N-terminal protected DOI from porcine insulin with a synthetic octapeptide corresponding to residues B-23-B30 of human insulin using trypsin as a catalyst.
However, this method is cumbersome in that it requires firstly a trypsin-catalyzed digestion of porcine insulin to form DOI, which is N-terminal protected by acylation with BOC-N.sub.3 and then incubated for 20 h with a separately synthesized human B23-B30 octapeptide, wherein B29 lysine is BOC-protected. The obtained (BOC).sub.3 -human insulin is subsequently deprotected with trifluoroacetic acid/anisole at 0.degree. C. for 60 min. The yield was 49% based on the (BOC).sub.2 --DOI used.
Similarly, Morihara et al. (Ref. 5) have synthesized human insulin from des-alanine (B-30)-insulin (DAI) obtained by digestion of porcine insulin with carboxypeptidase A for 8 h. The DAI (10 mM) was incubated with a large excess (0,5M) of threonine--OBu.sup.t ester at 37.degree. C. for 20 h in the presence of high concentrations of organic co-solvents. The formed [Thr--OBu.sup.t --B--30] insulin was then deprotected with trifluoroacetic acid in the presence of anisole. The yield was 41%. In a similar experiment [Thr--B--30] bovine insulin was obtained in 60% yield.
Also this method is cumbersome in that it requires a pretreatment of the initial insulin, long coupling times and a separate deprotection step. Also high amounts of organic co-solvents are necessary to minimize the hydrolytic activity of the enzyme.
In a similar experiment Morihara et al. (Ref. 6) used Achromobacter Protease I as enzymatic catalyst for the coupling of DAI with a large excess of Thr--OBu.sup.t under formation of [Thr--OBu.sup.t --B--30] insulin, which was isolated and deprotected as above. Although high yields (52%) may be obtained the reaction time was 20 h.
A similar experiment with bovine insulin leads to [Thr--OBu.sup.t --B--30] bovine insulin in 58% yield.
The processes disclosed in Ref. 5 and 6 are also described in European Patent Application No. EP 17938 published on Oct. 29, 1980 and Danish Application No. 1556/80.
Recently, it has been demonstrated that the enzyme carboxypeptidase-Y is an effective catalyst in peptide synthesis (Widmer and Johansen (Ref. 7) and Danish Patent Application No. 1443/79, filed Apr. 6, 1979). Furthermore, it has been shown that the enzyme under certain conditions catalyzes the exchange of the C-terminal amino acid in a peptide with another amino acid or amino acid derivative in a transpeptidation reaction (cf. International Appln. No. PCT/DK80/00020, filed Apr. 1, 1980 and published on Oct. 16, 1980 as WO 80/02151, and European Application No. EP 17485, published on Oct. 15, 1980, U.S. application Ser. No. 136,611 filed Apr. 2, 1980 and Ser. No. 220,022 filed Dec. 2, 1980 and based on the above PCT/DK80/0020. The underlying reaction principles are more fully explained by the inventors Breddam et al. (Ref. 11) who also discovered the so far unrecognized peptidyl-amino-acid-amide hydrolase activity of CPD-Y. The above-mentioned applications and the inventor's articles are incorporated herein by reference.
Although the general principle of an enzyme catalyzed transpeptidation reaction is thus described and exemplified in the above PCT and U.S. applications, the applicability thereof in connection with insulins has not been mentioned or shown.